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Vitamin A distribution and content in tissues of the lamprey Lampetra japonica.

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THE ANATOMICAL RECORD PART A 276A:134 –142 (2004)
Vitamin A Distribution and Content
in Tissues of the Lamprey,
Lampetra japonica
HEIDI L. WOLD,1 KENJIRO WAKE,2 NOBUYO HIGASHI,3 DAREN WANG,3
NAOSUKE KOJIMA,3 KATSUYUKI IMAI,3 RUNE BLOMHOFF,1 AND
HARUKI SENOO3*
1
Institute for Nutrition Research, Faculty of Medicine, University of Oslo,
Oslo, Norway
2
Department of Anatomy, Faculty of Medicine, Tokyo Medical and
Dental University, Tokyo, Japan
3
Department of Anatomy, Akita University School of Medicine, Akita, Japan
ABSTRACT
Vitamin A (retinol and retinyl ester) distribution and content in tissues of a lamprey (Lampetra japonica) were analyzed
by morphological methods, namely, gold chloride staining, fluorescence microscopy to detect specific vitamin A autofluorescence, and electron microscopy, as well as high-performance liquid chromatography (HPLC). Hepatic stellate cells showed
an abundance of vitamin A stored in lipid droplets in their cytoplasm. Similar cells storing vitamin A were present in the
intestine, kidney, gill, and heart in both female and male lampreys. Morphological data obtained by gold chloride staining
method, fluorescence microscopy, transmission electron microscopy, and HPLC quantification of retinol were consistent. The
highest level of total retinol measured by HPLC was found in the intestine. The second and third highest concentrations of
vitamin A were found in the liver and the kidney, respectively. These vitamin A-storing cells were not epithelial cells, but
mesoderm-derived cells. We propose as a hypothesis that these cells belong to the stellate cell system (family) that stores
vitamin A and regulates homeostasis of the vitamin in the whole body in the lamprey. Fibroblastic cells in the skin and
somatic muscle stored little vitamin A. These results indicate that there is difference in the vitamin A-storing capacity
between the splanchnic and intermediate mesoderm-derived cells (stellate cells) and somatic and dorsal mesoderm-derived
cells (fibroblasts) in the lamprey. Stellate cells derived from the splanchnic and intermediate mesoderm have high capacity
and fibroblasts derived from the somatic and dorsal mesoderm have low capacity for the storage of vitamin A in the lamprey.
Anat Rec Part A 276A:134 –142, 2004. © 2004 Wiley-Liss, Inc.
Key words: vitamin A; lamprey; stellate cell; gold chloride; fibroblast; mesoderm
Vitamin A is involved in many biological processes such
as cell growth, differentiation, morphogenesis, and apoptosis (Blomhoff, 1994). Homeostasis of vitamin A is rigidly controlled in the body (Blomhoff et al., 1990) with
hepatic stellate cells (vitamin A-storing cells, fat-storing
cells, interstitial cells, Ito cells) playing pivotal roles in its
regulation (Wake, 1980; Senoo et al., 1984, 1997, 1998;
Wake and Senoo, 1986; Blomhoff and Wake, 1991; Senoo
and Hata, 1994; Senoo, 2004).
In the liver of vertebrates, vitamin A (retinol and retinyl
esters) is stored in hepatic stellate cells that were first
described by von Kupffer (1876) and Rothe (1882), who
used the classical gold chloride method (Wake, 1964, 1971,
1974, 1980, 1982; Wake et al., 1986). The stellate cells are
distributed in the space between the sinusoidal endothelial cells and the hepatic parenchymal cells and contain
characteristically a number of lipid droplets that store
retinol in the form of retinyl esters (Higashi and Senoo,
2003; Sato et al., 2003). These lipid droplets emit retinol
autofluorescence under a fluorescence microscope and are
©
2004 WILEY-LISS, INC.
stained black by the gold chloride method. Storage of
retinol in these cells has been also confirmed by means of
Grant sponsor: Ministry of Education, Culture, Sports, Science,
and Technology of Japan; Grant numbers: Grant-in-Aid for Scientific Research (B) (2) (13470001), Grant-in-Aid for Scientific
Research (C) (11670001), Grant-in-Aid for Exploratory Research
(15659418).
Heidi L. Wold and Kenjiro Wake equally contributed to this study.
The present address of Kenjiro Wake: Liver Research Unit,
Minophagen Pharmaceutical Co. Ltd., No. 3 Tomizawa Bldg. 4F,
3-2-7 Yotsuya, Shinjuku-ku, Tokyo 160-0004, Japan.
*Correspondence to: Haruki Senoo, Department of Anatomy,
Akita University School of Medicine, 1-1-1 Hondo, Akita 010-8543,
Japan. Fax: ⫹81-18-834-7808. E-mail: senoo@ipc.akita-u.ac.jp
Received 18 September 2002; Accepted 2 September 2003
DOI 10.1002/ar.a.10345
VITAMIN A DISTRIBUTION AND CONTENT IN LAMPREY
3
radioautography after administration of H-retinol (Hirosawa and Yamada, 1973).
Stellate cells are distributed not only in the liver but
also in other organs (Kusumoto and Fujita, 1977; Wake,
1980; Nagy et al., 1997; Matano et al., 1999); however, the
exact distribution and the content of vitamin A in each
organ in the whole human body are still unknown.
It is of interest to know how organisms have developed
mechanisms to utilize, regulate, and store retinol during
vertebrate evolution. The lamprey is an intriguing model
for analyzing the evolution of retinol in the whole body of
a vertebrate, because due to its lowly position on the
taxonomic scale of vertebrates, and the fact that the lamprey can claim as its ancestors the primitive ostracoderms, the lamprey has been of interest to investigators
studying both ontogenetic and phylogenetic development
(Youson, 1985).
By the Carr-Price method a large amount of retinol was
demonstrated in the body of the adult lamprey (Lampetra
japonica) during its spawning migration (Higashi et al.,
1958). In addition, the appearance of retinol in lamprey
tissues was shown by Higashi and Yamada (1962) by use
of fluorescence microscopy.
The distribution, localization, and fine structure of the
stellate cells in the liver of adult lamprey were studied by
Youson et al. (1985, 1987), Peek et al. (1979), and Wake et
al. (1987). The stellate cells in the lamprey liver store
vitamin A as lipid droplets in their cytoplasm, but differ in
some of their properties from their counterpart in the
mammalian liver. These cells are responsible for periductal fibrosis during biliary atresia in the lamprey
(Yamamoto et al., 1986; Youson et al., 1987).
Pillar cells in gill filaments (Wake et al., 1989) and
mesangial cells in the renal corpuscle (Bauer and Wake,
1996) in the lamprey also store vitamin A.
However, the distribution and exact quantification and
qualification of vitamin A (retinol, retinyl esters with fatty
acids) in other tissues and organs in the whole body of the
lamprey have not yet been thoroughly investigated.
Therefore, to extend previous reports and analyze further
the distribution of stellate cells and the vitamin A content
in organs in the whole body and in serum of the lamprey,
we performed the present study by using high-performance liquid chromatography (HPLC).
MATERIALS AND METHODS
Adult lampreys (Lampetra japonica), classified as stage
VII in the life cycle of the anadromous parasitic lamprey
according to the criteria defined by Hardisty and Potter
(1971), were caught in rivers in Akita Prefecture, Japan,
in January and February 2000 –2002. A total of 11 animals (5 females and 6 males) were investigated in the
present study. The protocol for animal experimentation
described in this paper was previously approved by the
Animal Research Committee of Akita University School of
Medicine. All subsequent animal experiments adhered to
the Guidelines for Animal Experimentation of the university.
MORPHOLOGICAL METHODS
In total, seven lampreys (three females and four males)
were prepared for the morphological analysis. Four animals (two females and two males) were anesthetized in a
0.01% freshwater solution of meta-aminobezoic acid eth-
135
ylester methanesulfonate (MS222, Sankyo, Tokyo, Japan). After deep anesthetization, each organ (liver, kidney, heart, intestine, gill, somatic muscle, and skin) was
taken from the animals. The rest of the body was then
immediately frozen at – 80°C.
Gold Chloride Staining
Blocks of fresh organs from four lampreys (two females
and two males) were subjected to the gold chloride staining method modified by Wake (Wake, 1971; Wake et al.,
1986, 1987). Briefly, the tissue blocks were immersed in
0.05% chromic acid for about 2 hr at room temperature.
The blocks were then frozen and cut into 50-␮m-thick
sections and placed in 0.05% chromic acid for 10 min.
Then they were incubated in the gold chloride solution (1
ml of 1% gold chloride, 1 ml of 1% HCl, 98 ml of distilled
water) at 18 –20°C in total darkness for 17–20 hr. When
the sections had turned red-purple, they were dehydrated
with ethanol and mounted in balsam. The sections were
examined under a light microscope, and color photomicrographs were taken with Ektachrome ASA 100 film (Eastman Kodak). Also, monochromic photomicrographs were
taken by using Panatomic-X ASA 32 film (Eastman
Kodak).
Fluorescence Microscopy
Other tissue blocks were immersed in 3.7% formaldehyde for 24 hr at 4°C in total darkness, and 10-␮m-thick
sections were prepared with a freezing microtome as described previously (Wake, 1971; Senoo and Wake, 1985;
Wake et al., 1986; Nagy et al., 1997). The sections were
examined under a Zeiss Axioskop (excitation filter BP365/
12, barrier filter BP495/40) for the detection of the rapidly
fading green autofluorescence characteristic of vitamin A.
Color photomicrographs were taken by using Ektachrome
ASA 400 film (Eastman Kodak), and monochromic photomicrographs by using Tri-X Pan ASA 400 film (Eastman
Kodak).
Other tissue blocks fixed in 3.7% formaldehyde were
dehydrated in a graded ethanol series, embedded in paraffin, and sectioned. Sections were stained with hematoxylin and eosin, examined under a light microscope, and
photographed by using monochromatic Panatomic-X ASA
32 film (Eastman Kodak).
Transmission Electron Microscopy
The organs except the liver from three animals (one
female and two males) were perfused in situ for 1 or 2 min
via the heart with 1.5% glutaraldehyde and 0.062 M cacodylate buffer, pH 7.4, containing 1% sucrose. Part of the
excised liver was divided into blocks and perfused for 1 or
2 min by injection of the fixatives through branches of
blood vessels whose lumens appeared on the cut surface of
the blocks as described previously (Wake et al., 1987).
Under a dissection microscope the perfusion was performed with a syringe fitted with a needle (26 G ⫻ 1/2⬙).
The fixative consisted of 1.5% glutaraldehyde and 0.062 M
cacodylate buffer, pH 7.4, containing 1% sucrose. After the
perfusion fixation the organs were cut into small blocks,
and then the small tissue blocks were postfixed in 2%
osmium tetroxide in 0.1 M phosphate buffer, pH 7.4, for 2
hr at 4°C, dehydrated in a graded ethanol series, and
embedded in Poly/Bed 812. Ultrathin sections were cut on
an ultramicrotome and stained with 7% uranyl acetate
136
WOLD ET AL.
and 0.4% lead citrate. Thin sections were examined with a
JEOL-100 CX electron microscope (JEOL, Tokyo, Japan)
at an acceleration voltage of 100 kV. Thick sections were
examined under a light microscope after staining with 1%
toluidine blue containing 1% borax.
ANALYSIS OF RETINOL AND RETINYL
ESTERS BY HPLC
The content of vitamin A in serum (collected from the
incised gill), heart, liver, intestine, gonads, kidney, gill,
eye, brain, and somatic muscle obtained from the remaining four lampreys (two females and two males) was analyzed by HPLC. Both retinyl esters (palmitate, stearate,
oleate, and linoleate) and retinol were quantified.
Retinyl esters were extracted according to Barua et al.
(1993) with some minor modifications. Tissues (5–10 mg of
each) were homogenized in 90 ␮l of ice-cold phosphatebuffered saline (PBS) before the addition of 400 ␮l of
ice-cold 2-propanol/acetone (50:50 v/v). This mixture was
then vigorously shaken for 5 min and centrifuged for 15
min at 3,500 g at 10°C. An aliquot of 80 ␮l was injected
into an HPLC system equipped with a 250 ⫻ 4.6 mm C30
column from YMC (Midford, MA) and using an acetonitrile/dichloromethane (70:30 v/v) mobile phase delivered
at 2 ml/min (Furr et al., 1986). For serum analysis, 900 ␮l
of 2-propanol was added to 300 ␮l of serum. After shaking
and centrifugation as described above, an aliquot of 100 ␮l
was injected into an HPLC system equipped with a 250 ⫻
2.0 mm C18 column from Merck and the same mobile
phase as used for tissues delivered at 0.25 ml/min.
For retinol analyses, 50 mg of tissue was homogenized
in 450 ␮l of ice-cold PBS before the addition of 2,000 ␮l of
ice-cold 2-propanol containing all-trans-9-(4-methoxy2,3,6-trimethylphenyl)-3,7-dimethyl-2,4,6,8-nonatetraen1-ol (TMMP-retinol) as an internal standard and butylated hydroxytoluene (BHT) (10 mg/l) as an antioxidant
(for serum, 400 ␮l ⫹ 1,200 ␮l 2-propanol containing
TMMP-retinol and BHT). After shaking and centrifugation as described above, an aliquot of 1,000 ␮l was injected
into the HPLC system combining on-line solid-phase extraction and column switching (Gundersen and Blomhoff,
1998). Mobile phases M1 (2.2 ml/min), M2 (1 ml/min), and
M3 (0.5 ml/min) from pumps 1, 2, and 3 were 100% water,
100% methanol, and acetonitrile/water (85:15 v/v), respectively. Both retinyl esters and retinol were detected at 325
nm. All analyses were performed in duplicate from each
sample.
RESULTS
External Morphology
The head and associated portion of the adult lamprey
(Lampetra japonica) in lateral view showed the morphology typical of this species (Fig. 1).
Light and Electron Microscopy
Liver. In contrast to the larval lamprey (ammocoete)
and all other vertebrates, the adult lamprey does not have
a bile duct and the associated storage chamber, the gall
bladder (Sterling et al., 1967; Youson and Sidon, 1978;
Youson, 1981c; Sidon and Youson, 1983). The adult lamprey liver contains both parenchymal cells and nonparenchymal cells, namely, sinusoidal endothelial cells, Kupffer
cells, and hepatic stellate cells (vitamin A-storing cells)
(Youson et al., 1985). The stellate cells are located not only
perisinusoidally, but also perivascularly, and are also
found in the subcapsular dense connective tissue.
Under the fluorescence microscope, rapid-fading green
autofluorescence of vitamin A emanated from cells among
the liver parenchyma and within extensive areas of the
interstitial connective tissue (Fig. 2). The fluorescing cells
in the parenchyma were not parenchymal cells, but hepatic stellate cells. In the interstitial connective tissue the
fluorescing cells were fibloblast-like cells. The cell density
of fluorescing cells was higher in the connective tissue
than among the parenchymal cells. These fluorescing cells
were specifically stained black by the gold chloride method
(Fig. 3). Collagen fibers were stained red by this method.
Thus, the gold chloride method specifically stained the
vitamin A-storing cells black.
Gill. The lamprey gill is a respiratory organ. Blood
pumped by the heart flows through the ventral aorta into
afferent brachial arteries supplying capillaries in the gill
lamellae and then into the efferent brachial arteries,
which join to form the dorsal aorta (Randall, 1972). Only
the pillar cells in the gill emitted the autofluorescence of
vitamin A detected under the fluorescence microscope
(Fig. 4). These cells were regularly distributed in the gill
filaments. None of the epithelial cells lining the external
surface of the gill showed the vitamin A autofluorescence.
Kidney. The elements of the adult opisthonephroi are
confined within the wedge-shaped posterior portion of the
paired nephric folds, which extend into the coelomic cavity
from a dorsal mass of adipose tissue and pigment cells
(Youson, 1981b). The three major components of the opisthonephroi are the tubules, the renal corpuscle, and the
archinephric duct. The most conspicuous feature of the
adult opisthonephros, and a feature that distinguishes it
from the adult kidneys of other vertebrates, is the presence of a large, usually single, compound renal corpuscle
in each kidney. Each kidney is composed of a single glomus or network of capillaries that extends almost the
entire length of the kidney (Youson, 1981b). Throughout
the length of the renal corpuscle, tubules radiate out in a
semicircle from their origin at the nephric capsules. Each
tubule can be divided into several specific regions or segments that have distinct morphological features (Youson,
1981b). Mesangial cells within the renal corpuscle and
cells in the interstitium around the renal tubules emitted
vitamin A autofluorescence (Fig. 5); however, no epithelial
cells of the renal corpuscle or tubules showed the autofluorescence. The gold chloride method also demonstrated a
specific reaction of vitamin A in the connective tissue
around the renal tubules (Fig. 6), but not in the renal
epithelium. The fluorescing cells around the renal tubules
were identical with the cells stained black by the gold
chloride method.
An electron micrograph of a part of the renal corpuscle
showed the presence of vitamin A-lipid droplets in the
cytoplasm of mesangial cells (Fig. 7). Nuclei of the mesangial cells were deeply indented by the lipid droplets.
Neither podocytes nor endothelial cells in the renal corpuscle contained vitamin A-lipid droplets in their cytoplasm.
Cells in the interstitial tissue around the renal tubules
of the kidney contained vitamin A-lipid droplets in their
cytoplasm (Fig. 8). However, no epithelial cells of the renal
VITAMIN A DISTRIBUTION AND CONTENT IN LAMPREY
Fig. 1. Head and associated portion of a lamprey, Lampetra japonica, in lateral aspect. The lamprey was anesthetized as described in
Materials and Methods.
Fig. 2. Vitamin A autofluorescence released from stellate cells in the
liver parenchyma (arrow) and the interstitial connective tissue (CT). The
liver was prepared for fluorescence microscopy as described in Materials and Methods. Bar ⫽ 100 ␮m.
Fig. 3. Gold chloride staining of a lamprey liver that reduced gold
precipitates specifically in the stellate cells (arrows). Collagen fibers (CF)
are stained red. The liver was prepared for the gold chloride staining
method as described in Materials and Methods. Bar ⫽ 10 ␮m.
137
Fig. 4. Vitamin A autofluorescence emitted from clusters of lipid
droplets in pillar cells of a gill filament, as detected by fluorescence
microscopy. C, capillary; E, epithelium of a gill filament. Bar ⫽ 5 ␮m.
Fig. 5. Vitamin A autofluorescence is observed in the renal corpuscle
(G) and peritubular connective tissue in the kidney. RT, renal tubules.
Bar ⫽ 10 ␮m.
Fig. 6. Selectively black-stained cells are detected in the peritubular
connective tissue by the gold chloride staining method. RT, renal tubules. Bar ⫽ 10 ␮m.
138
WOLD ET AL.
Fig. 7. An electron micrograph of a part of the renal corpuscle of a
lamprey showing the presence of vitamin A-lipid droplets in the cytoplasm of mesangial cells (M). Nuclei of mesangial cells are deeply
indented by the lipid droplets. The kidney was prepared for transmission
electron microscopy as described in Materials and Methods. E, endothelial cell; P, podocyte. Bar ⫽ 5 ␮m.
Fig. 8. An electron micrograph showing cells storing lipid droplets
(arrows) in the peritubular connective tissue of the kidney. EP, epithelial
cells of the renal tubule. Bar ⫽ 5 ␮m.
Fig. 9. Vitamin A autofluorescence (arrows) is detected in the lamprey heart by fluorescence microscopy. M, heart muscular fiber. Bar ⫽
5 ␮m.
Fig. 10. A cell (SC) is observed by transmission electron microscopy
to contain a large vitamin A-lipid droplet (A) associated with collagen
fibers (CF) in the subendocardial connective tissue of the lamprey heart.
E, endothelium; M, heart muscle cell. Bar ⫽ 1 ␮m.
tubules stored vitamin A in their cytoplasmic lipid droplets.
cence of vitamin A (Fig. 9). These cells were fibroblast-like
cells with a morphology different from that of cardiac
muscle cells forming muscle fibers. These cells contained
one or two large vitamin A-lipid droplets (Fig. 10),
whereas the muscle cells contained myofibrils. No endothelial cells in the cardiac muscle contained vitamin Alipid droplets.
Heart. The heart of the lamprey consists of a sinus
venosus, atrium (auricle), ventricle, and bulbus arteriosus
(Fänge, 1972). Histological and electron microscopical
studies have shown striated myocardial fibers similar to
those of other vertebrates (Fänge, 1972; Hardisty and
Rovainen, 1982). Under the fluorescence microscope, cells
in the heart emitted the rapid-fading green autofluores-
Intestine. The intestine of all species of lampreys begins at its junction with the esophagus near the anterior
VITAMIN A DISTRIBUTION AND CONTENT IN LAMPREY
139
margin of the liver and is composed of anterior and posterior parts (Youson, 1981a). In the wall of the intestine, a
thin submucosa is sandwiched between the muscle and a
lamina propria mucosae (Youson, 1981a).
Intense vitamin A autofluorescence was detected in cells
of the lamina propria mucosae in the intestine (Fig. 11).
These cells were fibroblastic cells, and no autofluorescence
was demonstrated in the epithelial cells covering the lamina propria mucosae. The fluorescing cells reacted with
gold chloride and were stained black (data not shown).
Gold chloride-positive cells were demonstrated also in the
transverse section of the intestine showing a part of the
typhlosole (data not shown). Under the electron microscope these cells in the lamina propria mucosae that emitted autofluorescence of vitamin A and reacted with gold
chloride contained membrane-bound and non-membranebound lipid droplets (Wake, 1974) in their cytoplasm (Fig.
12).
Vitamin A autofluorescence was released also from the
layers of smooth muscle in the intestine (Fig. 13). These
cells reacted with gold chloride and were stained black
(data not shown), but they were different from the smooth
muscle cells.
Skin. The skin of vertebrates is composed of two primary layers, the epidermis and the dermis, which unite to
form the peripheral boundary separating the internal milieu of the individual from its external environment. While
the epidermis develops from ectodermal tissue and is organized as a stratified or pseudostratified epithelium, the
dermis is mesodermal in origin and is usually composed of
collagen fibers, pigment cells, and elements of the vascular and nervous systems. These dermal components are
often separated from the body wall musculature by several rows of fat cells, which constitute the subcutaneous
adipose layer (Lethbridge and Potter, 1981). The skin of
lampreys consists of epidermis, dermis, and subcutaneous
tissue (data not shown). The dermis contains layers of
flattened fibroblasts sandwiched between lamellae of collagen bundles. These fibroblasts as well as subcutaneous
fibroblasts contained no lipid droplets; no vitamin A was
demonstrated in these fibroblasts by either the gold chloride method or fluorescence microscopy (data not shown).
Somatic muscle. Striated muscles of the lamprey are
classified as somatic or visceral according to their embryological origins. The somatic muscles, including the extraocular, myotomal, and fin muscles, are derived from
the myomeres, whereas the visceral muscles, which are
used for feeding and ventilation, arise in the lateral plates
of the visceral arches in the head and gill regions (Hardisty and Rovainen, 1982). The myotomes constitute the
predominant muscle mass of the lamprey. They retain a
clear segmental organization and consist of short, longitudinally orientated striated muscle fibers, both in the
body and in the myotomal sheets that cover the visceral
muscles in the head and gill regions. The myotomes originate from the myomeres of the somites during embryological development and are thus termed somatic (Hardisty and Rovainen, 1982). Adipose cells loaded with fat
droplets in their cytoplasm were intercalated between
muscle fibers in the body muscle of the lamprey (data not
shown). In gold chloride-stained sections, a small number
of tiny gold chloride-reactive cells were interspersed between the muscle fibers (data not shown), but these cells
Fig. 11. Vitamin A autofluorescence is detected in cells of the lamina
propria (LP). E, epithelium. Bar ⫽ 5 ␮m.
Fig. 12. An electron micrograph showing membrane-bound (type I,
A1) and non-membrane-bound (type II, A2) lipid droplets (Wake, 1974) in
a cell (SC) in the lamina propria of the intestine. NF, unmyelinated nerve
fiber. Bar ⫽ 1 ␮m.
Fig. 13. Vitamin A autofluorescence released from cells in the muscle
layers of the intestine. Bar ⫽ 5 ␮m.
were different from the adipose cells. By elecron microscopy, small cells that appeared to be identical with the
gold chloride-reactive cells present between muscle fibers
contained lipid droplets in their cytoplasm (data not
140
WOLD ET AL.
TABLE 1. Vitamin A content in tissues of Lampetra japonica analyzed by high-performance liquid
chromatography concentration (nmol/g wet tissue)*
Female (n ⫽ 2, mean values)
Tissue
Serum
Heart
Liver
Intestine
Gonad
Kidney
Gill
Eye
Brain
Muscle
ROH⫹RE ROH
2
242
558
1687
52
441
278
92a
3a
41
RE
2
0
17
225
88
470
199 1488
10
42
10
431
98
181
28a
54
3a
1
4
7
Male (n ⫽ 2, mean values)
%16:0 %18:0 %18:1 %18:2 ROH⫹RE ROH
94
76
81
67
80
67
99
93
100
87
6
1
3
5
0
3
0
0
0
0
0
21
14
25
19
28
1
7
0
13
0
2
2
3
1
2
0
0
0
0
1
127
439
3006
66
231
84
39
6
35
1
7
28
301
31
7
29
10
4
3
RE
0
120
411
2705
35
224
56
29
2
32
%16:0 %18:0 %18:1 %18:2
98
81
85
65
74
64
62
93
100
84
2
0
2
6
0
2
0
0
0
0
0
18
11
26
26
33
37
7
0
16
0
1
2
2
0
1
1
0
0
0
*Retinol (ROH) and retinyl esters (RE) were measured as described in Materials and Methods. The results are presented as
mean values. n ⫽ 2 in each group.
a
n ⫽ 1. 16:0 ⫽ palmitate, 18:0 ⫽ stearate, 18:1 ⫽ oleate, 18:2 ⫽ linoleate.
shown), but the size and number of the lipid droplets were
small. It was hard to detect autofluorescence of vitamin A
in these cells under the fluorescence microscope.
Distribution of Vitamin A (Table 1)
The content of vitamin A (total retinol), retinol, retinyl
ester, and fatty acids (palmitate, stearate, oleate, and
linoleate) in various lamprey tissues is presented in Table 1.
The highest level of total retinol was found in the intestine. Of the total stored retinol, 88 –90% of it was retinyl
esters, mainly retinyl palmitate. The second highest concentration of vitamin A was found in the liver, and 84 –
94% of the total retinol was also in the form of retinyl
esters, mainly retinyl palmitate. The third highest level of
total retinol was found in the kidney, where 97–98% of it
was also retinyl esters, again mainly retinyl palmitate.
The levels of total retinol in the heart and gill were also
high, but the levels in the somatic muscle and gonads were
low, and the levels in the serum and brain were very low.
The percentage of retinyl esters to total retinol and the
compositions of the four fatty acids, namely, palmitate,
stearate, oleate, and linoleate, were essentially the same
in both female and male organs examined.
Essentially no difference existed in total retinol concentration in each corresponding organ (female liver and
male liver, for instance) between female and male lampreys.
DISCUSSION
The Stellate Cell System Storing Retinol
In mammals, hepatic stellate cells store 80% of the total
retinol in the whole body as retinyl esters in the lipid
droplets in their cytoplasm (Wake, 1971, 1980; Blomhoff
et al., 1990; Imai and Senoo, 1998; Imai et al., 2000).
Several papers reported that cells in other organs such as
kidney, intestine, and pancreas of rats, mice, and humans
can store retinol and contribute to the regulation of vitamin A homeostasis (Hirosawa and Yamada, 1973; Wake et
al., 1987; Nagy et al., 1997; Matano et al., 1999; Apte et
al., 1998; Bachem et al., 1998).
To investigate systematically the distribution of cells
storing vitamin A in adult lamprey body and to compare
this distribution with that of the hepatic stellate cells, we
performed this study. Cells responsible for storage of vitamin A were distributed in all the visceral organs examined, namely, intestine, liver, kidney, gill, and heart. The
storage cells were not epithelial cells, but mesenchymal
cells, and they resembled hepatic stellate cells from the
viewpoint of morphology; these cells emitted the autofluorescence of vitamin A, reacted with gold chloride, and fine
structure analysis revealed lipid droplets in their cytoplasm.
The pillar cells, namely, the lining cells of the blood
lacunae in the secondary lamellae of the gill filaments,
have been already described as vitamin A-storing cells of
gill filaments (Wake et al., 1987, 1989). We found lipid
droplets containing vitamin A in these cells, which findings are consistent with these earlier data.
Morphological analysis of the kidney indicated that
mesangial cells in the renal corpuscle and mesenchymal
cells in the interstitium had characteristics of the stellate
cell (vitamin A-storing cell).
From these data we propose as a hypothesis that the
stellate cell system or family exists in splanchnic and
intermediate mesoderm-derived tissues in the whole body
of the lamprey (Fig. 14).
Centralization of the Storage Site for Vitamin A
during Evolution
Lampreys and hagfish are the only extant jawless fish
or agnathans. They may have shared a common ancestor
in the early Cambrian period, but they diverged very early
in their evolutionary histories. Lampreys have had a
rather conserved evolution since they first appeared 350
million years ago (Youson and Al-Mahrouki, 1999).
In mammals, vitamin A is mainly stored in the liver;
especially hepatic stellate cells store 80% of the vitamin of
the whole body. However, vitamin A in the lamprey was
found to be localized not only in the hepatic stellate cells,
but also in other mesenchymal cells derived from the
mesoderm. As mentioned above, we propose a hypothesis
that the stellate cell system exists in lampreys. Our
present data suggest that the storage site of vitamin A
changed from the wide distribution in the splanchnic and
intermediate mesoderm-derived tissues in lampreys to the
liver in mammals during evolution. This phenomenon
VITAMIN A DISTRIBUTION AND CONTENT IN LAMPREY
141
Fig. 14. Distribution of vitamin A in the stellate cell system in the lamprey, Lampetra japonica. Stellate
cells (vitamin A-storing cells), derived from the splanchnic and intermediate mesoderm (intestine, liver,
kidney, heart, gill), have high capacity; and fibroblasts, derived from the somatic and dorsal mesoderm (skin,
somatic muscle), have low capacity for the storage of vitamin A.
might be called centralization of the storage site of vitamin A during evolution.
Agreement between Morphological and HPLC
Analytical Data
Morphological data obtained by the gold chloride staining method, fluorescence microscopy, and transmission
electron microscopy, and data on retinol quantified by
HPLC were consistent. The high concentration of vitamin
A in the intestine detected by HPLC was consistent with
the presence of stellate cells that contained large vitamin
A-lipid droplets in the intestine. Also, the high concentration of total retinol in the liver found by HPLC was consistent with stellate cells that contained large vitamin
A-lipid droplets in hepatic parenchyma and interstitial
connective tissue. Thus, the HPLC analysis supported the
morphological findings.
Differentiation of Mesodermal Cells
We defined the mesenchymal cells containing multiple
vitamin A-lipid droplets or a single large vitamin A-lipid
droplet as stellate cells, and the mesenchymal cells having
no or a single small vitamin A-lipid droplet in their cytoplasm as fibroblasts. Cells belonging to the stellate cell
system in the intestine, liver, kidney, heart, and gill in the
lamprey stored a large amount of vitamin A. On the other
hand, fibroblasts in the skin and somatic muscle stored
little vitamin A. These results indicate that the stellate
cells, derived from the splanchnic and intermediate mesoderm, have high capacity, and that the fibroblasts, derived
from the somatic and dorsal mesoderm, have no or low
capacity for the storage of vitamin A. Thus, from the
viewpoint of vitamin A storage, there is differentiation of
function in mesoderm-derived cells according to their localization in the body (Fig. 14).
In conclusion, this is the first report of systematic analysis of the stellate cell system in the lamprey, which has
shown the difference in vitamin A storage between the
stellate cells derived from the splanchnic and intermediate mesoderm and fibroblasts derived from the somatic
and dorsal mesoderm.
ACKNOWLEDGMENTS
The authors thank Dr. Mitsuru Sato (Department of
Anatomy, Akita University School of Medicine) for his
valuable discussions. Expert technical support by Mitsutaka Miura (Department of Anatomy, Akita University
School of Medicine) is also highly appreciated.
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